Laura Nigi

MicroRNAs: Novel Players in the Dialogue between Pancreatic Islets and Immune System in Autoimmune Diabetes.

MicroRNAs are small noncoding RNA molecules that regulate gene expression in all cell types. Therefore, these tiny noncoding RNA molecules are involved in a wide range of biological processes, exerting functional effects at cellular, tissue, and organ level. In pancreatic islets of Langerhans, including beta-cells, microRNAs are involved in cell differentiation as well as in insulin secretion, while in immune cells they have been shown to play pivotal roles in development, activation, and response to antigens. Indeed, it is not surprising that microRNA alterations can lead to the development of several diseases, including type 1 diabetes (T1D). Type 1 diabetes is the result of a selective autoimmune destruction of insulin-producing beta-cells, characterized by islet inflammation (insulitis), which leads to chronic hyperglycemia. Given the growing importance of microRNA in the pathophysiology of T1D, the aim of this review is to summarize the most recent data on the potential involvement of microRNAs in autoimmune diabetes. Specifically, we will focus on three different aspects: (i) microRNAs as regulators of immune homeostasis in autoimmune diabetes; (ii) microRNA expression in pancreatic islet inflammation; (iii) microRNAs as players in the dialogue between the immune system and pancreatic endocrine cells.

Circulating microRNAs and diabetes mellitus: a novel tool for disease prediction, diagnosis, and staging?

Diabetes is a complex, multifactorial group of metabolic diseases characterized by chronic hyperglycaemia due to pancreatic beta-cell dysfunction and/or loss. It is characterized by an asymptomatic and highly variable prodromic phase, which renders diabetes mellitus difficult to be predicted with sufficient accuracy. Despite several efforts in the identification and standardization of newly trustable. Biomarkers able to predict and follow-up diabetes and to specifically subtype its different forms, few of them have proven of clinical utility. Recently, a new class of endogenous non-coding small RNAs, namely microRNAs, have been indicated as putative biomarkers, being released by cells and tissues and found in a cell-free circulating form in many biological fluids, including serum and/or plasma. MicroRNAs have been initially identified as promising biomarkers in cancer, and nowadays their application has been extended to other diseases, including diabetes. Although an increasing number of studies focused on the evaluation of circulating microRNAs in diabetes, few reproducibly identified microRNAs as biomarkers for disease prediction or follow-up. Technological problems as well as the need to obtain highly standardized operating procedures and methods are still an issue in such research field. In this review, we comprehensively resume the main and most recent findings on circulating microRNAs, and their possible use as biomarkers to predict and follow-up diabetes and its complications, as well as the methodological challenges to standardize accurate operating procedures for their analysis.

MicroRNA expression profiles of human iPSCs differentiation into insulin-producing cells.

AimsMicroRNAs are a class of small noncoding RNAs, which control gene expression by inhibition of mRNA translation. MicroRNAs are involved in the control of biological processes including cell differentiation. Here, we aim at characterizing microRNA expression profiles during differentiation of human induced pluripotent stem cells (hiPSCs) into insulin-producing cells.MethodsWe differentiated hiPSCs toward endocrine pancreatic lineage following a 18-day protocol. We analyzed genes and microRNA expression levels using RT real-time PCR and TaqMan microRNA arrays followed by bioinformatic functional analysis.ResultsMicroRNA expression profiles analysis of undifferentiated hiPSCs during pancreatic differentiation revealed that 347/768 microRNAs were expressed at least in one time point of all samples. We observed 18 microRNAs differentially expressed: 11 were upregulated (miR-9-5p, miR-9-3p, miR-10a, miR-99a-3p, miR-124a, miR-135a, miR-138, miR-149, miR-211, miR-342-3p and miR-375) and 7 downregulated (miR-31, miR-127, miR-143, miR-302c-3p, miR-373, miR-518b and miR-520c-3p) during differentiation into insulin-producing cells. Selected microRNAs were further evaluated during differentiation of Sendai-virus-reprogrammed hiPSCs using an improved endocrine pancreatic beta cell derivation protocol and, moreover, in differentiated NKX6.1+ sorted cells. Following Targetscan7.0 analysis of target genes of differentially expressed microRNAs and gene ontology classification, we found that such target genes belong to categories of major significance in pancreas organogenesis and development or exocytosis.ConclusionsWe detected a specific hiPSCs microRNAs signature during differentiation into insulin-producing cells and demonstrated that differentially expressed microRNAs target several genes involved in pancreas organogenesis.

The case for virus-induced type 1 diabetes.

Purpose of review Type 1 diabetes (T1D) results from the immune-mediated destruction of pancreatic insulin-producing cells because of the interaction among genetic susceptibility, the immune system and environmental factor(s). A possible role of viral infections in T1D pathogenesis has been hypothesized for some time; however, only in the most recent years, studies performed at the molecular and cellular level are starting to shed light on this issue. Recent findings Studies in animal models and in man have shown that viruses can indeed infect pancreatic beta-cells, inducing islet inflammation and functional damage. In addition, recent in-situ investigations performed on pancreatic tissue samples have provided evidence that in addition to adaptive immune response, innate immunity is involved in T1D pathogenesis and the whole pancreas (not only its endocrine portion) is infiltrated by immune-mediated phenomena. Summary The established role of inflammation in the insulitic process and the increasing evidence in support of the contribution of viral infections to a proinflammatory islet scenario are strongly suggestive that viruses may indeed contribute to beta-cell damage and dysfunction, thus setting the stage for the design of antiviral strategies (e.g. vaccines and antiviral drugs) aimed at protecting the beta-cells.

Combination therapy with metformin plus vildagliptin in type 2 diabetes mellitus

Introduction: Type 2 diabetes mellitus (T2DM) is pathophysiologically characterized by a combination of insulin resistance and beta-cell dysfunction. Consequently, a proper treatment of such a disease should target both of these defects. Dipeptidyl peptidase-4 (DPP-4) inhibitors are among the most recent additions to the therapeutic options for T2DM and are able to increase circulating levels of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), thus stimulating glucose-dependent insulin secretion. Areas covered: This paper provides an overview of the clinical results of combination therapy with metformin and the DPP-4 inhibitor vildagliptin in T2DM patients. Expert opinion: Vildagliptin–metformin single-tablet combination is indicated for the treatment of T2DM patients not achieving a sufficient glycemic control at their maximally tolerated dose of metformin. Results from clinical trials provide evidence of vildagliptin efficacy administered in addition to metformin, as either first- or second-line treatment. The vildagliptin–metformin association seems to have favorable effects on beta-cell function and is characterized by good safety and tolerability profiles when compared with other antidiabetic agents. Of note, data available suggest that administration of fixed-dose combination products, together with the low incidence of adverse gastrointestinal events, may improve compliance and adherence of patients to therapy, resulting in an improved metabolic control.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors

Islet-reactive CD8+ T cells are common in the periphery, but home to the pancreas preferentially in the context of type 1 diabetes. At home in the pancreas Type 1 diabetes (T1D) is associated with enrichment of autoreactive CD8+ T cells that target destruction of pancreatic islets. Culina et al. studied islet-reactive CD8+ T cells reactive to the zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes in healthy individuals and T1D patients, which showed similar functionality and similar frequencies and naïve phenotypes in the peripheral circulation across both groups. In contrast, ZnT8186–194-reactive CD8+ T cells were enriched in the pancreas of T1D patients relative to healthy controls and showed cross-reactivity to an epitope from the commensal Bacteroides stercoris. These results indicate that incomplete central tolerance may allow the survival of these islet-reactive CD8+ T cells in the periphery, and that proinflammatory conditions in the islets can contribute to T1D progression. The human leukocyte antigen–A2 (HLA-A2)–restricted zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186–194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186–194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186–194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186–194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186–194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Regulatory T-cells from pancreatic lymphnodes of patients with type-1 diabetes express increased levels of microRNA miR-125a-5p that limits CCR2 expression

Autoimmune type 1 diabetes (T1D) is thought to be caused by a defective immune regulation with regulatory T (Treg) cells playing a fundamental role in this process. Tolerance mechanisms depend on tunable responses that are sensitive to minor perturbations in the expression of molecules that can be carried out by multiple epigenetic mechanisms, including regulation by microRNAs. In this study, microRNA expression profile was investigated in Treg cells isolated from peripheral blood (PB) and from pancreatic draining lymph nodes (PLN) of T1D patients and non-diabetic subjects. Among 72 microRNAs analyzed, miR-125a-5p resulted specifically hyper-expressed in Treg cells purified from PLN of T1D patients. TNFR2 and CCR2 were identified as miR-125a-5p target genes. Elevated miR-125a-5p was detected in Treg cells isolated from PLN but not from PB of donors with T1D and was associated with reduced CCR2 expression. A specific beta-cell expression of the CCR2-ligand (CCL2) was observed in the pancreata of cadaveric donors, suggesting that beta-cells are prone to attract CCR2+ Treg cells. These novel data propose a mechanism, occurring in PLNs of T1D patients, involving increased expression of miR-125a-5p on Treg cells which results into reduced expression of CCR2, thus limiting their migration and eventual function in the pancreas.

Towards an Earlier and Timely Diagnosis of Type 1 Diabetes: Is it Time to Change Criteria to Define Disease Onset?

Type 1 diabetes (T1D) is the immune-mediated form of diabetes requiring insulin treatment and affecting both children and adults. The incidence of T1D is increasing dramatically and has doubled in the past 2 decades. In the recent years, significant knowledge on the disease natural history has been gained and, nowadays, diabetes-related autoantibodies make T1D a predictable disease. Despite this great advance in the field of T1D, we still use diagnostic criteria defined by the American Diabetes Association (ADA) in 1997. In other autoimmune endocrine disorders (e.g., Hashimoto’s thyroiditis and Addison’s disease), that share several features with T1D, diagnosis is made early in the presence of circulating autoantibodies together with subclinical thyroid/adrenal functional impairment and treatments are often provided in the absence of a frank clinical glandular insufficiency. With this review, we propose to anticipate diagnosis also in T1D at the stage in which subjects have circulating multiple islet autoantibodies, are dysglycemic but are still insulin independent. We believe that anticipating T1D diagnosis can lead to better disease management and prevention of secondary complications but can also provide the possibility to perform earlier and likely more effective interventions for a disease that to date has proven controllable but still incurable.

Prospective Validation of ATA and ETA Sonographic Pattern Risk of Thyroid Nodules Selected for FNAC

Context Recently, the American Thyroid Association (ATA) and the European Thyroid Association (ETA) have proposed that thyroid ultrasound (US) should be used to stratify the risk of malignancy in thyroid nodules and to aid decision-making about whether fine-needle aspiration cytology (FNAC) is indicated. Objective To validate and to compare the ATA and ETA US risk stratification systems of thyroid nodules in a prospective series of thyroid nodules submitted to FNAC. Setting We prospectively evaluated 432 thyroid nodules selected for FNAC from 340 patients. Cytology reports were based on the five categories according to the criteria of the British Thyroid Association. Results The proportion of Thy2 nodules decreased significantly, whereas the proportion of Thy4/Thy5 nodules significantly increased with increasing US risk class (P < 0.0001). The ability to identify benign and malignant nodules was similar between ATA and ETA systems. According to ATA and ETA US risk stratification systems, 23.7% and 56.0% nodules did not meet the criteria for FNAC, respectively. Considering only categories at lower risk of malignancy, the cumulative malignancy rate in these nodules was 1.2% for ATA and 1.7% for ETA US risk stratification systems. Conclusions ETA and ATA US risk stratification systems provide effective malignancy risk stratification for thyroid nodules. In clinical practice, using this approach, we should be able to reduce the number of unnecessary FNAC without losing clinically relevant thyroid cancer.

Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells

Aims/hypothesisThe Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells.MethodsFormalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets.ResultsAn isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking.Conclusions/interpretationThe SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.